Campus Units
Biochemistry, Biophysics and Molecular Biology, Roy J. Carver Department of, Chemistry
Document Type
Article
Publication Version
Accepted Manuscript
Publication Date
3-2020
Journal or Book Title
Protein Expression and Purification
Volume
167
First Page
105540
DOI
10.1016/j.pep.2019.105540
Abstract
Various fusion tags are commonly employed to increase the heterologous expression and solubility of aggregation-prone proteins within Escherichia coli. Herein, we present a protocol for efficient recombinant expression and purification of the human RNA demethylases Alkbh5 and FTO. Our method incorporates a novel fusion tag (the N-terminal domain of bacterial enzyme I, EIN) that dramatically increases the solubility of its fusion partner and is promptly removed upon digestion with a protease. The presented protocol allows for the production of mg amounts of Alkbh5 and FTO in 1L of both rich and minimal media. We developed a liquid chromatography-mass spectrometry (LC-MS)-based assay to confirm that both proteins are enzymatically active. Furthermore, the LC-MS method developed here is applicable to other members of the AlkB family of Fe(II)/α-ketoglutarate-dependent dioxygenases. The superior protein yield, afforded by our expression and purification method, will facilitate biochemical investigations into the biological function of the human RNA demethylases and endorse employment of EIN as a broadly applicable fusion tag for recombinant expression projects.
Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 4.0 International License.
Copyright Owner
Elsevier Inc.
Copyright Date
2019
Language
en
File Format
application/pdf
Recommended Citation
Khatiwada, Balabhadra; Purslow, Jeffrey; Underbakke, Eric S.; and Venditti, Vincenzo, "N-terminal fusion of the N-terminal domain of bacterial enzyme I facilitates recombinant expression and purification of the human RNA demethylases FTO and Alkbh5" (2020). Chemistry Publications. 1185.
https://lib.dr.iastate.edu/chem_pubs/1185
Comments
This is a manuscript of an article published as Khatiwada, Balabhadra, Jeffrey A. Purslow, Eric S. Underbakke, and Vincenzo Venditti. "N-terminal fusion of the N-terminal domain of bacterial enzyme I facilitates recombinant expression and purification of the human RNA demethylases FTO and Alkbh5." Protein Expression and Purification (2019): 105540. DOI: 10.1016/j.pep.2019.105540. Posted with permission.